Methods and compositions for treating hepatitis B

ABSTRACT

The invention provides compositions comprising a plurality of yeast cells, wherein said plurality of yeast cells are characterized by their ability to normalize the level of serum glutamate-pyruvate Transaminase (GPT), or reduce serum HBsAg levels in a subject, said ability resulting from their having been cultured in the presence of an alternating electric field having a specific frequency and a specific field strength. Also provided are methods of making and using these compositions.

FIELD OF THE INVENTION

The invention relates to compositions that can ameliorate or preventhepatitis B and are useful as a dietary supplement or medication. Thesecompositions contain yeast cells obtainable by growth in electromagneticfields with specific frequencies and field strengths.

BACKGROUND OF THE INVENTION

Hepatitis is caused by viruses, bacteria, substance abuse, certainmedicines, or serious structural damages to the liver. Most commonly,hepatitis is caused by one of three viruses: hepatitis A virus,hepatitis B virus, or hepatitis C virus. Hepatitis B, also called “serumhepatitis,” is caused by hepatitis B virus (HBV). HBV spreads throughinfected body fluids. Most hepatitis B patients recover from theirillness completely within six months. However, some patients go on todevelop chronic hepatitis and liver cirrhosis. These patients becomelifelong carriers of HBV and can spread the virus to other people.

Hepatitis is a serious public health problem. It is estimated that thereare over 350 million hepatitis B carriers worldwide, representing 5% ofthe world population. It is also estimated that 10 to 30 million peoplebecome infected with the virus every year. At present, the drug commonlyused in the treatment of chronic hepatitis B is interferon. Thistreatment, however, does not work for everyone with chronic hepatitis B,and can cause strong side effects, such as flu-like symptoms, rashes,and depression. There remains a need for an effective method to treathepatitis B.

SUMMARY OF THE INVENTION

This invention is based on the discovery that certain yeast cells can beactivated by electromagnetic fields having specific frequencies andfield strengths to produce substances beneficial for the liver.Compositions comprising these activated yeast cells can be used as adietary supplement or medication for treating liver diseases, e.g., foralleviating or preventing hepatitis B.

This invention embraces a composition comprising a plurality of yeastcells that have been cultured in an alternating electric field having afrequency in the range of about 7900-12400 MHz (e.g., 7900-8100,9850-10050, or 12200-12400 MHz), and a field intensity in the range ofabout 240-500 mV/cm (e.g., 260-280, 270-290, 290-320, 300-330, 310-340,320-350, 330-360, 360-390, 400-440, or 430-470 mV/cm). The yeast cellsare cultured in the alternating electric field for a period of timesufficient to substantially increase the capability of said plurality ofyeast cells to produce substances beneficial for the liver (e.g., fortreating hepatitis B). In one embodiment, the frequency and/or the fieldstrength of the alternating electric field can be altered within theaforementioned ranges during said period of time. In other words, theyeast cells can be exposed to a series of electromagnetic fields. Anexemplary period of time is about 40-160 hours (e.g., 60-145 hours).

Also included in this invention is a composition comprising a pluralityof yeast cells that have been cultured under acidic conditions in analternating electric field having a frequency in the range of about9850-12400 MHz (e.g., 12200-12400 MHz) and a field strength in the rangeof about 270 to 420 mV/cm (e.g., 300-330 or 360-390 mV/cm). In oneembodiment, the yeast cells are exposed to a series of electromagneticfields. An exemplary period of time is about 40-110 hours (e.g., 58-78hours).

Included in this invention are also methods for making the abovecompositions.

Yeast cells that can be included in this composition can be derived fromparent strains publically available from the China GeneralMicrobiological Culture Collection Center (“CGMCC”), China Committee forCulture Collection of Microorganisms, Institute of Microbiology, ChineseAcademy of Sciences, Haidian, P.O. BOX 2714, Beijing, 100080, China.Useful yeast species include, but are not limited to Saccharomycescerevisiae, Saccharomyces carlsbergensis, Saccharomyces rouxii,Saccharomyces sake, Saccharomyces uvarum, Saccharomyces sp.,Schizosaccharomyces pombe, and Rhodotorula aurantiaca. For instance, theyeast cells can be of the strain Saccharomyces cerevisiae Hansen AS2.561or AS2.69, Saccharomyces sp. AS2.311, Schizosaccharomyces pombe LindnerAS2.994, Saccharomyces sake Yabe ACCC2045, Saccharomyces uvarum BeijerIFFI1044, Saccharomyces rouxii Boutroux AS2.180, Saccharomycescerevisiae Hansen Var. ellipsoideus AS2.612, Saccharomycescarlsbergensis Hansen AS2.377, or Rhodotorula rubar (Demme) LodderAS2.282. Other useful yeast strains are illustrated in Table 1.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Exemplary methods and materialsare described below, although methods and materials similar orequivalent to those described herein can also be used in the practice ortesting of the present invention. All publications and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intended to be limiting. Throughout this specification and claims,the word “comprise,” or variations such as “comprises” or “comprising”will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing an exemplary apparatus foractivating yeast cells using electromagnetic fields. 1: yeast culture;2: container; 3: power supply.

FIG. 2 is a schematic diagram showing an exemplary apparatus for makingyeast compositions of the invention. The apparatus comprises a signalgenerator (such as models 83721B and 83741A manufactured by HP) andinterconnected containers A, B and C.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on the discovery that certain yeast strains canbe activated by electromagnetic fields (“EMF”) having specificfrequencies and field strengths to produce agents useful in treatingliver diseases, e.g., hepatitis B. Yeast compositions containingactivated yeast cells can be used as medication, or as a dietarysupplement in the form of health drinks or dietary pills.

Since the activated yeast cells contained in these yeast compositionshave been cultured to endure acidic conditions (pH 2.5-4.2), thecompositions are stable in the stomach and can pass on to theintestines. Once in the intestines, the yeast cells are ruptured byvarious digestive enzymes, and the bioactive agents are released andreadily absorbed.

I. Yeast Strains Useful in the Invention

The types of yeasts useful in this invention include, but are notlimited to, yeasts of the genera of Saccharomyces, Rhodotorula, andSchizosaccharomyces.

Exemplary species within the above-listed genera include, but are notlimited to, the species illustrated in Table 1. Yeast strains useful inthis invention can be obtained from laboratory cultures, or frompublically accessible culture depositories, such as CGMCC and theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209. Non-limiting examples of useful strains (with theaccession numbers of CGMCC) are Saccharomyces cerevisiae Hansen AS2.561and AS2.69, Saccharomyces sp. AS2.311, Schizosaccharomyces pombe LindnerAS2.994, Saccharomyces sake Yabe ACCC2045, Saccharomyces uvarum BeijerIFFI1044, Saccharomyces rouxii Boutroux AS2.180, Saccharomycescerevisiae Hansen Var. ellipsoideus AS2.612, Saccharomycescarlsbergensis Hansen AS2.377, and Rhodotorula rubar (Demme) LodderAS2.282. Other non-limiting examples of useful strains are listed inTable 1. In general, preferred yeast strains in this invention are thoseused for fermentation in the food and wine industries. As a result,compositions containing these yeast cells are safe for humanconsumption.

The preparation of the yeast compositions of this invention is notlimited to starting with a pure strain of yeast. A yeast composition ofthe invention may be produced by culturing a mixture of yeast cells ofdifferent species or strains.

TABLE 1 Exemplary Yeast Strains Saccharomyces cerevisiae Hansen ACCC2034ACCC2035 ACCC2036 ACCC2037 ACCC2038 ACCC2039 ACCC2040 ACCC2041 ACCC2042AS2. 1 AS2.4 AS2.11 AS2.14 AS2.16 AS2.56 AS2.69 AS2.70 AS2.93 AS2.98AS2.101 AS2.109 AS2.110 AS2.112 AS2.139 AS2.173 AS2.174 AS2.182 AS2.196AS2.242 AS2.336 AS2.346 AS2.369 AS2.374 AS2.375 AS2.379 AS2.380 AS2.382AS2.390 AS2.393 AS2.395 AS2.396 AS2.397 AS2.398 AS2.399 AS2.400 AS2.406AS2.408 AS2.409 AS2.413 AS2.414 AS2.415 AS2.416 AS2.422 AS2.423 AS2.430AS2.431 AS2.432 AS2.451 AS2.452 AS2.453 AS2.458 AS2.460 AS2.463 AS2.467AS2.486 AS2.501 AS2.502 AS2.503 AS2.504 AS2.516 AS2.535 AS2.536 AS2.558AS2.560 AS2.561 AS2.562 AS2.576 AS2.593 AS2.594 AS2.614 AS2.620 AS2.628AS2.631 AS2.666 AS2.982 AS2.1190 AS2.1364 AS2. 1396 IFFI1001 IFFI1002IFFI1005 IFFI1006 IFFI1008 IFFI1009 IFFI1010 IFFI1012 IFFI1021 IFFI1027IFFI1037 IFFI1042 IFFI1043 IFFI1045 IFFI1048 IFFI1049 IFFI1050 IFFI1052IFFI1059 IFFI1060 IFFI1062 IFFI1063 IFFI1202 IFFI1203 IFFI1206 IFFI1209IFFI1210 IFFI1211 IFFI1212 IFFI1213 IFFI1214 IFFI1215 IFFI1220 IFFI1221IFFI1224 IFFI1247 IFFI1248 IFFI1251 IFFI1270 IFFI1277 IFFI1287 IFFI1289IFFI1290 IFFI1291 IFFI1292 IFFI1293 IFFI1297 IFFI1300 IFFI1301 IFFI1302IFFI1307 IFFI1308 IFFI1309 IFFI1310 IFFI1311 IFFI1331 IFFI1335 IFFI1336IFFI1337 IFFI1338 IFFI1339 IFFI1340 IFFI1345 IFFI1348 IFFI1396 IFFI1397IFFI1399 IFFI1411 IFFI1413 IFFI1441 IFFI1443 Saccharomyces cerevisiaeHansen Var. ellipsoideus (Hansen) Dekker ACCC2043 AS2.2 AS2.3 AS2.8AS2.53 AS2.163 AS2.168 AS2.483 AS2.541 AS2.559 AS2.606 AS2.607 AS2.611AS2.612 Saccharomyces chevalieri Guilliermond AS2.131 AS2.213Saccharomyces delbrueckii AS2.285 Saccharomyces delbrueckii Lindner ver.mongolicus (Saito) Lodder et van Rij AS2.209 AS2.1157 Saccharomycesexiguous Hansen AS2.349 AS2.1158 Saccharomyces fermentaii (Saito) Lodderet van Rij AS2.286 AS2.343 Saccharomyces logos van laer et Denamur exJorgensen AS2.156 AS2.327 AS2.335 Saccharomyces mellis (Fabian etQuinet) Lodder et kreger van Rij AS2.195 Saccharomyces mellisMicroellipsoides Osterwalder AS2.699 Saccharomyces oviformis OsteralderAS2.100 Saccharomyces rosei (Guilliermond) Lodder et Kreger van RijAS2.287 Saccharomyces rouxii Boutroux AS2.178 AS2.180 AS2.370 AS2.371Saccharomyces sake Yabe ACCC2045 Candida arborea AS2.566 Candida lambica(Lindner et Genoud) van. Uden et Buckley AS2.1182 Candida krusei(Castellani) Berkhout AS2.1045 Candida lipolytica (Harrison) Diddens etLodder AS2.1207 AS2.1216 AS2.1220 AS2.1379 AS2.1398 AS2.1399 AS2.1400Candida parapsilosis (Ashford) Langeron et Talice Var. intermedia VanRij et Verona AS2.491 Candida parapsilosis (Ashford) Langeron et TaliceAS2.590 Candida pulcherrima (Lindner) Windisch AS2.492 Candida rugousa(Anderson) Diddens et Lodder AS2.511 AS2.1367 AS2.1369 AS2.1372 AS2.1373AS2.1377 AS2.1378 AS2.1384 Candida tropicalis (Castellani) BerkhoutACCC2004 ACCC2005 ACCC2006 AS2.164 AS2.402 AS2.564 AS2.565 AS2.567AS2.568 AS2.617 AS2.637 AS2.1387 AS2.1397 Candida utilis HennebergLodder et Kreger Van Rij AS2.120 AS2.281 AS2.1180 Crebrothecium ashbyii(Guillermond) Routein (Eremothecium ashbyii Guilliermond) AS2.481AS2.482 AS2.1197 Geotrichum candidum Link ACCC2016 AS2.361 AS2.498AS2.616 AS2.1035 AS2.1062 AS2.1080 AS2.1132 AS2.1175 AS2.1183 Hansenulaanomala (Hansen)H et P sydow ACCC2018 AS2.294 AS2.295 AS2.296 AS2.297AS2.298 AS2.299 AS2.300 AS2.302 AS2.338 AS2.339 AS2.340 AS2.341 AS2.470AS2.592 AS2.641 AS2.642 AS2.782 AS2.635 AS2.794 Hansenula arabitolgensFang AS2.887 Hansenula jadinii (A. et R Sartory Weill et Meyer)Wickerham ACCC2019 Hansenula saturnus (Klocker) H et P sydow ACCC2020Hansenula schneggii (Weber) Dekker AS2.304 Hansenula subpelliculosaBedford AS2.740 AS2.760 AS2.761 AS2.770 AS2.783 A52.790 AS2.798 AS2.866Kloeckera apiculata (Reess emend. Klocker) Janke ACCC2022 ACCC2023AS2.197 AS2.496 AS2.714 ACCC2021 AS2.711 Lipomycess starkeyi Lodder etvan Rij AS2.1390 ACCC2024 Pichia farinosa (Lindner) Hansen ACCC2025ACCC2026 AS2.86 AS2.87 AS2.705 AS2.803 Pichia membranaefaciens HansenACCC2027 AS2.89 AS2.661 AS2.1039 Rhodosporidium toruloides BannoACCC2028 Rhodotorula glutinis (Fresenius) Harrison AS2.2029 AS2.280ACCC2030 AS2.102 AS2.107 AS2.278 AS2.499 AS2.694 AS2.703 AS2.704AS2.1146 Rhodotorula minuta (Saito) Harrison AS2.277 Rhodotorula rubar(Demme) Lodder AS2.21 AS2.22 AS2.103 AS2.105 AS2.108 AS2.140 AS2.166AS2.167 AS2.272 AS2.279 AS2.282 ACCC2031 Rhodotorula aurantiaca (Saito)Lodder AS2.102 AS2.107 AS2.278 AS2.499 AS2.694 AS2.703 AS2.1146Saccharomyces carlsbergensis Hansen AS2.113 ACCC2032 ACCC2033 AS2.312AS2.116 AS2.118 AS2.121 AS2.132 AS2.162 AS2.189 Saccharomyces uvarumBeijer IFFI1023 IFFI1032 IFFI1036 IFFI1044 IFFI1072 IFFI1205 IFFI1207Saccharomyces willianus Saccardo AS2.5 AS2.7 AS2.119 AS2.152 AS2.293AS2.381 AS2.392 AS2.434 AS2.614 AS2.1189 Saccharomyces sp. AS2.311Saccharomycodes ludwigii Hansen ACCC2044 AS2.243 AS2.508 Saccharomycodessinenses Yue AS2.1395 Schizosaccharomyces octosporus Beijerinck ACCC2046AS2.1148 Schizosaccharomyces pombe Lindner ACCC2047 ACCC2048 AS2.214AS2.248 AS2.249 AS2.255 AS2.257 AS2.259 AS2.260 AS2.274 AS2.994 AS2.1043AS2.1149 AS2.1178 IFFI1056 Sporobolomyces roseus Kluyver et van NielACCC2049 ACCC2050 AS2.19 AS2.962 AS2.1036 ACCC2051 AS2.261 AS2.262Torulopsis candida (Saito) Lodder AS2.270 ACCC2052 Torulopsis famta(Harrison) Lodder et van Rij ACCC2053 AS2.685 Torulopsis globosa (Olsonet Hammer) Lodder et van Rij ACCC2054 AS2.202 Torulopsis inconspicuaLodder et Kreger van Rij AS2.75 Trichosporon behrendii Lodder et Kregervan Rij ACCC2056 AS2.1193 Trichosporon capitatum Diddens et LodderACCC2056 AS2.1385 Trichosporon cutaneum (de Reurm et al.) Ota ACCC2057AS2.25 AS2.570 AS2.571 AS2.1374 Wickerhamia fluorescens (Soneda) SonedaACCC2058 AS2.1388II. Application of Electromagnetic Fields

An electromagnetic field useful in this invention can be generated andapplied by various means well known in the art. For instance, the EMFcan be generated by applying an alternating electric field or anoscillating magnetic field.

Alternating electric fields can be applied to cell cultures throughelectrodes in direct contact with the culture medium, or throughelectromagnetic induction. See, e.g., FIG. 1. Relatively high electricfields in the medium can be generated using a method in which theelectrodes are in contact with the medium. Care must be taken to preventelectrolysis at the electrodes from introducing undesired ions into theculture and to prevent contact resistance, bubbles, or other features ofelectrolysis from dropping the field level below that intended.Electrodes should be matched to their environment, for example, usingAg—AgCl electrodes in solutions rich in chloride ions, and run at as lowa voltage as possible. For general review, see Goodman et al., Effectsof EMF on Molecules and Cells, International Review of Cytology, ASurvey of Cell Biology, Vol. 158, Academic Press, 1995.

The EMFs useful in this invention can also be generated by applying anoscillating magnetic field. An oscillating magnetic field can begenerated by oscillating electric currents going through Helmholtzcoils. Such a magnetic field in turn induces an electric field.

The frequencies of EMFs useful in this invention range from about 7900MHz to 12400 MHz (e.g., 7900-8100, 9850-10050, or 12200-12400 MHz).Exemplary frequencies include 7986, 8009, 9949, 12293, and 12312 MHz.The field strength of the electric field useful in this invention rangesfrom about 240-500 mV/cm (e.g., 260-280, 270-290, 290-320, 300-330,310-340, 320-350, 330-360, 360-390, 400-440, or 430-470 mV/cm).Exemplary field strengths include 267, 272, 285, 298, 315, 317, 327,337, 347, 375, 416, and 446 mV/cm.

When a series of EMFs are applied to a yeast culture, the yeast culturecan remain in the same container while the same set of EMF generator andemitters is used to change the frequency and/or field strength. The EMFsin the series can each have a different frequency or a different fieldstrength; or a different frequency and a different field strength. Suchfrequencies and field strengths are preferably within theabove-described ranges. Although any practical number of EMFs can beused in a series, it may be preferred that the yeast culture be exposedto a total of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more EMFs in aseries. In one embodiment, the yeast culture is exposed to a series ofEMFs, wherein the frequency of the electric field is alternated in therange of about 7900-8100, 9850-10050, and 12200-12400 MHz.

Although the yeast cells can be activated after even a few hours ofculturing in the presence of an EMF, it may be preferred that theactivated yeast cells be allowed to multiply and grow in the presence ofthe EMF(s) for a total of 40-160 hours.

FIG. 1 illustrates an exemplary apparatus for generating alternatingelectric fields. An electric field of a desired frequency and intensitycan be generated by an AC source (3) capable of generating analternating electric field, preferably in a sinusoidal wave form, in thefrequency range of 5 to 20,000 MHz. Signal generators capable ofgenerating signals with a narrower frequency range can also be used. Ifdesired, a signal amplifier can also be used to increase the output. Theculture container (2) can be made from a non-conductive material, e.g.,glass, plastic or ceramic. The cable connecting the culture container(2) and the signal generator (3) is preferably a high frequency coaxialcable with a transmission frequency of at least 30 GHz.

The alternating electric field can be applied to the culture by avariety of means, including placing the yeast culture (1) in closeproximity to the signal emitters such as a metal wire or tube capable oftransmitting EMFs. The metal wire or tube can be made of red copper, andbe placed inside the container (2), reaching as deep as 3-30 cm. Forexample, if the fluid in the container (2) has a depth of 15-20 cm,20-30 cm, 30-50 cm, 50-70 cm, 70-100 cm, 100-150 cm or 150-200 cm, themetal wire can be 3-5 cm, 5-7 cm, 7-10 cm, 10-15 cm, 15-20 cm, 20-30 cm,and 25-30 cm from the bottom of the container (2), respectively. Thenumber of metal wires/tubes used can be from 1 to 10 (e.g., 2 to 3). Itis recommended, though not mandated, that for a culture having a volumeup to 10 L, metal wires/tubes having a diameter of 0.5 to 2 mm be used.For a culture having a volume of 10-100 L, metal wires/tubes having adiameter of 3 to 5 mm can be used. For a culture having a volume of100-1000 L, metal wires/tubes having a diameter of 6 to 15 nun can beused. For a culture having a volume greater than 1000 L, metalwires/tubes having a diameter of 20-25 mm can be used.

In one embodiment, the electric field is applied by electrodes submergedin the culture (1). In this embodiment, one of the electrodes can be ametal plate placed on the bottom of the container (2), and the otherelectrode can comprise a plurality of electrode wires evenly distributedin the culture (1) so as to achieve even distribution of the electricfield energy.

III. Culture Media

Culture media useful in this invention contain sources of nutrients thatcan be assimilated by yeast cells. Complex carbon-containing substancesin a suitable form (e.g., carbohydrates such as sucrose, glucose,dextrose, maltose, xylose, cellulose, starch, etc.) can be the carbonsources for yeast cells. The exact quantity of the carbon sources can beadjusted in accordance with the other ingredients of the medium. Ingeneral, the amount of carbohydrates varies between about 1% and 10% byweight of the medium and preferably between about 1% and 5%, and mostpreferably about 2%. These carbon sources can be used individually or incombination. Amino acid-containing substances such as beef extract andpeptone can also be added. In general, the amount of amino acidcontaining substances varies between about 0.1% and 1% by weight of themedium and preferably between about 0.1% and 0.5%. Among the inorganicsalts which can be added to a culture medium are the customary saltscapable of yielding sodium, potassium, calcium, phosphate, sulfate,carbonate, and like ions. Non-limiting examples of nutrient inorganicsalts are (NH₄)₂HPO₄, CaCO₃, KH₂PO₄, K₂ HPO₄, MgSO₄, NaCl, and CaSO₄.

IV. Electromagnetic Activation of Yeast Cells

To activate or enhance the ability of yeast cells to produce agentsuseful for treating live diseases (e.g., hepatitis B), these cells canbe cultured in an appropriate medium under sterile conditions at 20-35°C. (e.g., 28-32° C.) for a sufficient amount of time (e.g., 60-145hours) in an alternating electric field or a series of alternatingelectric fields as described above.

An exemplary set-up of the culture process is depicted in FIG. 1 (seeabove). An exemplary culture medium contains the following per 1000 mlof sterile water: 18 g of mannitol, 50 μg of Vitamin B₆, 50 μg ofVitamin B₁₂, 50 μg of Vitamin B₃, 100 □g of Vitamin H, 35 ml of fetalbovine serum, 0.2 g of KH₂PO₄, 0.25 g of MgSO₄.7H₂O, 0.3 g of NaCl, 0.2g of CaSO₄.2H₂O, 4 g of CaCO₃.5H₂O, and 2.5 g of peptone. Yeast cells ofthe desired strain(s) are then added to the culture medium to form amixture containing 1×10⁸ cells per 1000 ml of culture medium. The yeastcells can be of any of the strains listed in Table 1. The mixture isthen added to the apparatus shown in FIG. 1.

The activation process of the yeast cells involves the following steps:(1) maintaining the temperature of the activation apparatus at 24-33° C.(e.g., 28-32° C.), and culturing the yeast cells for 24-30 hours (e.g.,28 hours); (2) applying an alternating electric field having a frequencyof 7986 MHz and a field strength of 260-280 mV/cm (e.g., 267 mV/cm) for11-17 hours (e.g., 15 hours); (3) then applying an alternating electricfield having a frequency of 8009 MHz and a field strength of 310-340mV/cm (e.g., 315 mV/cm) for 32-38 hours (e.g., 36 hours); (4) thenapplying an alternating electric field having a frequency of 9949 MHzand a field strength of 320-350 mV/cm (e.g., 337 mV/cm) for 38-44 hours(e.g., 42 hours); (5) then applying an alternating electric field havinga frequency of 12293 MHz and a field strength of 330-360 mV/cm (e.g.,347 mV/cm) for 35-41 hours (e.g., 39 hours); and (6) then applying analternating electric field having a frequency of 12312 MHz and a fieldstrength of 260-280 mV/cm (e.g., 272 mV/cm) for 6-12 hours (e.g., 10hours). The activated yeast cells are then recovered from the culturemedium by various methods known in the art, dried (e.g., bylyophilization) and stored at 4° C. Preferably, the concentration of thedried yeast cells is no less than 10¹⁰ cells/g.

V. Acclimatization of Yeast Cells to the Gastric Environment

Because the yeast compositions of this invention must pass through thestomach before reaching the small intestine, where the effectivecomponents are released from these yeast cells, it is preferred thatthese yeast cells be cultured under acidic conditions to acclimatize thecells to the gastric juice. This acclimatization process results inbetter viability of the yeast cells in the acidic gastric environment.

To achieve this, the yeast powder containing activated yeast cells canbe mixed with a highly acidic acclimatizing culture medium at 10 g(containing more than 10¹⁰ activated cells per gram) per 1000 ml. Theyeast mixture is then cultured first in the presence of an alternatingelectric field having a frequency of 12293 MHz and a field strength of360-390 mV/cm (e.g., 375 mV/cm) at about 28 to 32° C. for 42 to 50 hours(e.g., 46 hours). The resultant yeast cells can then be furtherincubated in the presence of an alternating electric field having afrequency of 12312 MHz and a field strength of 300-330 mV/cm (e.g., 317mV/cm) at about 28 to 32° C. for 16 to 28 hours (e.g., 20 hours). Theresulting acclimatized yeast cells are then dried and stored either inpowder form (≧10¹⁰ cells/g) at room temperature or in vacuum at 0-4° C.

An exemplary acclimatizing culture medium is made by mixing 700 ml freshpig gastric juice and 300 ml wild Chinese hawthorn extract. The pH ofthe acclimatizing culture medium is adjusted to 2.5 with 0.1 Mhydrochloric acid (HCl) and 0.2 M potassium hydrogen phthalate(C₆H₄(COOK)COOH). The fresh pig gastric juice is prepared as follows. Atabout 4 months of age, newborn Holland white pigs are sacrificed, andthe entire contents of their stomachs are retrieved and mixed with 2000ml of water under sterile conditions. The mixture is then allowed tostand for 6 hours at 4° C. under sterile conditions to precipitate fooddebris. The supernatant is collected for use in the acclimatizingculture medium. To prepare the wild Chinese hawthorn extract, 500 g offresh wild Chinese hawthorn is dried under sterile conditions to reducewater content (≦8%). The dried fruit is then ground (≧20 mesh) and addedto 1500 ml of sterilized water. The hawthorn slurry is allowed to standfor 6 hours at 4° C. under sterile conditions. The hawthorn supernatantis collected to be used in the acclimatizing culture medium.

VI. Manufacture of Yeast Compositions

To manufacture the yeast compositions of the invention, an apparatusdepicted in FIG. 2 or an equivalent thereof can be used. This apparatusincludes three containers, a first container (A), a second container(B), and a third container (C), each equipped with a pair of electrodes(4). One of the electrodes is a metal plate placed on the bottom of thecontainers, and the other electrode comprises a plurality of electrodewires evenly distributed in the space within the container to achieveeven distribution of the electric field energy. All three pairs ofelectrodes are connected to a common signal generator.

The culture medium used for this purpose is a mixed fruit extractsolution containing the following ingredients per 1000 L: 300 L of wildChinese hawthorn extract, 300 L of jujube extract, 300 L of Schisandrachinensis (Turez) Baill seed extract, and 100 L of soy bean extract. Toprepare hawthorn, jujube and Schisandra chinensis (Turez) Baill seedextracts, the fresh fruits are washed and dried under sterile conditionsto reduce the water content to no higher than 8%. One hundred kilogramsof the dried fruits are then ground (≧20 mesh) and added to 400 L ofsterilized water. The mixtures are stirred under sterile conditions atroom temperature for twelve hours, and then centrifuged at 1000 rpm toremove insoluble residues. To make the soy bean extract, fresh soy beansare washed and dried under sterile conditions to reduce the watercontent to no higher than 8%. Thirty kilograms of dried soy beans arethen ground into particles of no smaller than 20 mesh, and added to 130L of sterilized water. The mixture is stirred under sterile conditionsat room temperature for twelve hours and centrifuged at 1000 rpm toremove insoluble residues. To make the culture medium, these ingredientsare mixed according to the above recipe, and the mixture is autoclavedat 121° C. for 30 minutes and cooled to below 40° C. before use.

One thousand grams of the activated yeast powder prepared as describedabove (Section V, supra) is added to 1000 L of the mixed fruit extractsolution, and the yeast solution is transferred to the first container(A) shown in FIG. 2. The yeast cells are then cultured in the presenceof an alternating electric field having a frequency of 12293 MHz and afield strength of about 400-440 mV/cm (e.g., 416 mV/cm) at 28-32° C.under sterile conditions for 32 hours. The yeast cells are furtherincubated in an alternating electric field having a frequency of 12312MHz and a field strength of 290-320 mV/cm (e.g., 298 mV/cm). Theculturing continues for another 12 hours.

The yeast culture is then transferred from the first container (A) tothe second container (B) which contains 1000 L of culture medium (ifneed be, a new batch of yeast culture can be started in the nowavailable first container (A)), and subjected to an alternating electricfield having a frequency of 12293 MHz and a field strength of 430-470mV/cm (e.g., 446 mV/cm) for 24 hours. Subsequently the frequency andfield strength of the electric field are changed to 12312 MHz and260-280 mV/cm (e.g., 272 mV/cm), respectively. The culturing continuesfor another 12 hours.

The yeast culture is then transferred from the second container (B) tothe third container (C) which contains 1000 L of culture medium, andsubjected to an alternating electric field having a frequency of 12293MHz and a field strength of 310-340 mV/cm (e.g., 327 mV/cm) for 24hours. Subsequently the frequency and field strength of the electricfield are changed to 12312 MHz and 270-290 mV/cm (e.g., 285 mV/cm),respectively. The culturing continues for another 12 hours.

The yeast culture from the third container (C) can then be packaged intovacuum sealed bottles for use as a dietary supplement, e.g., healthdrinks, or medication in the form of pills, powder, etc. If desired, thefinal yeast culture can also be dried within 24 hours and stored inpowder form. The dietary supplement can be taken three to four timesdaily at 30-60 ml per dose for a three-month period, preferably 10-30minutes before meals and at bedtime.

In some embodiments, the compositions of the invention can also beadministered intravenously or peritoneally in the form of a sterileinjectable preparation. Such a sterile preparation can be prepared asfollows. A sterilized health drink composition is first treated underultrasound (20,000 Hz) for 10 minutes and then centrifuged for another10 minutes. The resulting supernatant is adjusted to pH 7.2-7.4 using 1M NaOH and subsequently filtered through a membrane (0.22 μm forintravenous injection and 0.45 μM for peritoneal injection) understerile conditions. The resulting sterile preparation is submerged in a35-38° C. water bath for 30 minutes before use. In other embodiments,the compositions of the invention may also be formulated withpharmaceutically acceptable carriers to be orally administered in anyorally acceptable dosage form including, but not limited to, capsules,tablets, suspensions or solutions.

The yeast compositions of the present invention are derived from yeastsused in food and pharmaceutical industries. The yeast compositions arethus devoid of side effects associated with many pharmaceuticalcompounds.

VII. EXAMPLES

The following examples are meant to illustrate the methods and materialsof the present invention. Suitable modifications and adaptations of thedescribed conditions and parameters which are obvious to those skilledin the art are within the spirit and scope of the present invention.

The activated yeast compositions used in the following experiments wereprepared as described above, using Saccharomyces cerevisiae HansenAS2.561 cells cultured in the presence of an alternating electric fieldhaving the electric field frequency and field strength exemplified inthe parentheses following the recommended ranges listed in Section IV,supra. Control yeast compositions were those prepared in the same mannerexcept that the yeast cells were cultured in the absence of EMFs. Unlessotherwise indicated, the yeast compositions and the correspondingcontrols were administered to the animals by intragastric feeding.

Example 1 Effects of Yeast Compositions against HBsAg

The HBV surface coat is composed of hepatitis B surface antigens(“HBsAg”). HBsAg is produced in larger quantities than required for thevirus to reproduce. The excess surface antigens clump into sphericalparticles or form rods of variable length. These spherical particles canalso encapsulate a core particle and produce a complete and infectiousviral particle that enters the blood stream and infects other livercells. The excess spheres, rods and complete viral particles enter theblood stream in large numbers and are easily detectable.HbsAg-positivity is the current standard used to indicate HBV infection.The presence of HBsAg for more than six months is generally taken toindicate chronic infection.

In this experiment, the effectiveness of the activated yeast compositionin reducing HBsAg level was assessed using an ELISA assay.

Preparation of Yeast Compositions for ELISA:

Under sterile conditions, a bottle (100 ml/bottle, about 10⁸ cells/ml)of the activated yeast composition (AY) was mixed with 100 ml ofde-ionized H₂O in a 200 ml beaker and the mixture incubated at 28-30° C.for two hours. The mixture was then sonicated at 3000 Hz for 15 minutesand centrifuged at 1000 rpm for 10 minutes. The supernatant was thenfiltered through a 0.45 μm membrane. De-ionized water was added to bringthe volume to 100 ml. The pH of the solution was adjusted to 7.0 with0.1 M NaOH and HCl, and then stored at 4° C. Before use, three differentconcentrations of the solution were prepared: 1×50 μl (stock solutionwithout further concentration), 2×50 μl (100 μl of stock solutionconcentrated to 50 μl), and 3×50 μl (150 μl of stock solutionconcentrated to 50 μl). Control yeast composition solutions (NY) wereprepared in the same way.

Preparation of HBsAg Solutions:

HBsAg was purified from HBsAg positive serum (with a titer of 1:8) usingcellulose ion-exchange affinity chromatography. The preparation wasstored in aliquots at 4° C. Two HBsAg concentrations were used for thisexperiment: P/N (Positive/Negative)=10.92, and P/N=14.26. P/N is theratio between the HBsAg concentration of an HbsAg-positive serum andthat of an HbsAg-negative serum.

Experimental Procedure:

Six solutions were prepared by mixing 50 μl of the yeast compositionsolutions at three different concentrations with 50 μl of the HBsAgpreparations at two different concentrations. HBsAg positive (where noyeast composition solution was added) and HBsAg negative (no HBsAg)controls, as well as a blank control (where H₂O was used in lieu ofyeast composition and HBsAg) were also included. These mixtures wereincubated at 37° C. for four hours. The ELISA plate were coated with 100μl of purified hepatitis B surface antibody (“HbsAb”) per well at 4° C.for 48 hours. The plates were then washed several times with wash bufferand spun dry. The yeast composition solution-HBsAg mixtures and thevarious controls were each added to a HBsAb-coated well and incubated at43° C. for two hours. The plates were washed several times and spun dry,and 100 μl of HRP-HBsAb (1:100) in 10% fetal bovine serum was added perwell and incubated at 43° C. for one hour. The plates were then washedseveral times and spun dry. 100 μl of o-phenylenediamine-hydrogenperoxide was added per well. After incubation at 37° C. in the dark for30 minutes, the reactions were stopped by adding 50 μl of 2 M H₂SO₄ perwell. The optical density of the samples was measured at 492 nm, usingthe blank sample for calibration. The P/N values of the reactions werecalculated based on the average OD values (i.e., OD value for thesamples divided by the OD value of the negative control). The data areshown in Table 2 below.

TABLE 2 P/N when HBsAg P/N = 14.26 P/N when HBsAg P/N = 10.92 1X50 2X503X50 1X50 2X50 3X50 Group (50 μl) (50 μl) (50 μl) (50 μl) (50 μl) (50μl) AY 0.95 0.44 0.18 0.42 0.19 0.11 NY 14.11 14.03 13.96 10.86 10.6310.34

The data demonstrate that the activated yeast composition significantlyreduced the level of HBsAg (P/N<0.95) compared to the control yeastcomposition (P/N>10.34). By general medical standards, a P/N value of<1.2 indicates significant effect of treatment; a P/N value of <2.1,average effect; a P/N value of 3.8-4.25, low effect; and a P/N valueof >4.25, no effect.

Example 2 Effects of Yeast Compositions on Glutamate-PyruvateTransaminase Activity

Glutamate-pyruvate transaminase (GPT) normally is expressed inhepatocytes. When the liver tissue undergoes necrosis or is otherwisedamaged, GPT is released into the blood stream, elevating the level ofserum GPT. Thus, the serum GPT level is one of the important indicatorsof liver functions.

To evaluate the effects of the activated yeast composition of thisinvention on serum GPT activity, the yeast compositions were tested inpatients with chronic hepatitis B (either Chronic Persistent Hepatitis Bor Chronic Active Hepatitis B). The study was conducted under thedirection of physicians.

In this study, the patients with Chronic Persistent Hepatitis B orChronic Active Hepatitis B (these two groups of patients were studiedseparately) were randomly divided into three groups, namely AY (fortreatment with the activated yeast composition), NY (for treatment withthe control yeast composition), CK (positive control group, fortreatment with Stronger Neominophagen C, or SNMC, a known drug fortreating hepatitis B). The AY group patients were each given 30 ml ofthe activated yeast composition (about 10⁸ cells/ml), three times dailyfor six months. The NY patients were treated in the same manner exceptthat they were given the control yeast composition, in lieu of theactivated yeast composition. The CK patients were each given 40 ml ofSNMC (1.0 mg/ml) via intraveinous injection daily for six months.

At the end of the sixth month, blood samples were taken from eachpatient to determine the serum GPT level. To do so, 0.1 ml of serum fromeach paitent was mixed with 0.5 ml of the glutamate-pyruvate substratesolution (1 M) and incubated in a 37° C. water bath for 30 minutes. Then0.5 ml of 2,4-dinitrophenylhydrazine was added and the incubationcontinued for another 20 minutes. Finally, 5 ml of 0.4 M NaOH was added.The control reaction was prepared in the same manner except that theserum was added immediately after, not before, the 30 minute incubationstep. The optical density of the sample was measured at 520 nm, usingthe control reaction for calibration. The GPT concentration wasdetermined using a standard curve. Data in Table 3 below show that thenumber of patients in each group whose serum GPT level returned tonormal after treatment.

TABLE 3 Patients with Patients with Chronic Persistent Chronic ActiveHepatitis B Hepatitis B After Treatment After Treatment # of % of # of %of Patients Patients Patients Patients Total # with with Total # withwith of Normal Normal of Normal Normal Group Patients [GPT] [GPT]Patients [GPT] [GPT] AY 22 19 86.3 26 22 84.6 NY 23 0 0 25 0 0 CK 20 315.0 27 4 14.8

The data demonstrate that the activated yeast composition significantlyrestored serum GPT to normal levels in patients with chronic hepatitisB, and was superior to SNMC in doing so.

While a number of embodiments of this invention have been set forth, itis apparent that the basic constructions may be altered to provide otherembodiments which utilize the compositions and methods of thisinvention.

1. A composition comprising a plurality of yeast cells, wherein saidplurality of yeast cells are characterized by their ability to normalizethe level of serum glutamate-pyruvate Transaminase (GPT), or reduceserum HBsAg levels in a subject, said ability resulting from theirhaving been cultured in the presence of an alternating electric fieldhaving a frequency in the range of 7900-12400 MHz and a field strengthin the range of 240-500 mV/cm, as compared to yeast cells not havingbeen so cultured.
 2. The composition of claim 1, wherein said frequencyis in the range of 7900-8100, 9850-10050, or 12200-12400 MHz.
 3. Thecomposition of claim 1, wherein said field strength is in the range of260-280, 270-290, 290-320, 300-330, 310-340, 320-350, 330-360, 360-390,400-440, or 430-470 mV/cm.
 4. The composition of claim 1, wherein saidyeast cells are of the species selected from the group consisting ofSaccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomycesrouxii, Saccharomyces sake, Saccharomyces uvarum, Saccharomyces sp.,Schizosaccharomyces pombe, and Rhodotorula aurantiaca.
 5. Thecomposition of claim 1, wherein said yeast cells are of the straindeposited at the China General Microbiological Culture Collection Centerwith an accession number selected from the group consisting ofSaccharomyces cerevisiae Hansen AS2.561 and AS2.69, Saccharomyces sp.AS2.311, Schizosaccharomyces pombe Lindner AS2.994, Saccharomyces sakeYabe ACCC2045, Saccharomyces uvarum Beijer IFFI1044, Saccharomycesrouxii Boutroux AS2.180, Saccharomyces cerevisiae Hansen Var.ellipsoideus AS2.612, Saccharomyces carlsbergensis Hansen AS2.377, orRhodotorula rubar (Demme) Lodder AS2.282.
 6. The composition of claim 1,wherein said composition is in the form of a tablet, powder, or a healthdrink.
 7. The composition of claim 1, wherein said composition is in theform of a health drink.
 8. A method of treating hepatitis B in asubject, comprising administering the composition of claim 1 to thesubject.
 9. The method of claim 8, comprising oral administration.
 10. Amethod of preparing a yeast composition, comprising culturing aplurality of yeast cells in the presence of an alternating electricfield having a frequency in the range of 7900-12400 MHZ and a fieldstrength in the range of 240-500 mV/cm for a period of time sufficientto substantially increase the capability of said plurality of yeastcells to normalize the level of serum glutamate-pyruvate Transaminase orreduce serum Hepatitis B surface antigen levels as compared to yeastcells not having been so cultured.